PTEN/MMAC1 mutations in hepatocellular carcinomas Ya Juan Yao 1 , Xiao Li Ping 1 , Hong Zhang 1 , Fei Fei Chen 1 , Patricia K Lee 1 , Habibul Ahsan 3 , Chien-Jen Chen 4 , Po-Huang Lee 5 , Monica Peacocke 1 , Regina M Santella 2 and Hui C Tsou* ,1 1 Department of Dermatology, School of Public Health, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA; 2 Division of Environmental Health Science, School of Public Health, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA; 3 Division of Epidemiology, School of Public Health, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA; 4 Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan; 5 Department of Surgery, College of Public Health, National Taiwan University, Taipei, Taiwan Mutations in the PTEN/MMAC1 gene have been identi®ed in several types of human cancers and cancer cell lines, including brain, endometrial, prostate, breast, thyroid, and melanoma. In this study, we screened a total of 96 hepatocellular carcinoma (HCC) samples from Taiwan, where HCC is the leading cancer in males and third leading cancer in females, for mutations in the PTEN/MMAC1 gene. Complete sequence analysis of these samples demonstrated a missense mutation in exon 5 (K144I) and exon 7 (V255A) from HCC samples B6- 21 and B6-2, respectively. A putative splice site mutation was also detected in intron 3 from sample B6-2. Both B6-21 and B6-2 were previously shown to contain missense mutations in the coding sequences of the p53 gene. Functional studies with the two missense mutations demonstrated that while mutation V255A in exon 7 resulted in a loss of phosphatase activity, mutation K144I in exon 5 retained its phosphatase activity. Additionally, we identi®ed a silent mutation (P96P) in exon 5 of the PTEN/MMAC1 gene from HCC sample B6-22. These data provide the ®rst evidence that the PTEN/MMAC1 gene is mutated in a subset of HCC samples. Keywords: PTEN/MMAC1; mutation; liver; tumor Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world (Montesano et al., 1997). The incidence of HCC varies in its geographic distribution. It has the highest incidence in Qidong, China and sub-Saharan, Africa (Chen et al., 1997), however, it is relatively rare in North America and Europe. The increased incidence of HCC in China and Africa is mainly caused by the high level of dietary a¯atoxin B 1 (AFB 1 ) exposure, and the high incidence of chronic hepatitis B virus (HBV) infection (Peers et al., 1976; Yeh et al., 1989). Genetic analysis of 26 HCC samples from these two areas revealed a high incidence of an AG G?AG T transversional changes in codon 249 of the p53 gene (Hsu et al., 1991; Bressac et al., 1991). By contrast, p53 mutations in HCC samples from low AFB 1 exposure areas occurred less frequently and were not con®ned to cordon 249 (Buetow et al., 1992; Aguilar et al., 1994). In addition to p53, other tumor suppressor genes have also been implicated in HCC. A loss of heterozygosity (LOH) of the retinoblastoma (RB) and APC genes was also detected in HCC samples from China (44 and 7% respectively) (Fujimoto et al., 1994). Recently, a series of somatic mutations in the coding sequence of the b-catenin gene was identi®ed in 18% (14/75) of HCC samples screened (Miyoshi et al., 1998). Activation of proto-oncogenes, such as c-myc and cyclin D1, by DNA ampli®cation has also been observed in a subset of HCC samples (Peng et al., 1993; Zhang et al., 1993; Nishida et al., 1994). PTEN/MMAC1/TEP1, a tumor suppressor gene, was ®rst isolated from cell lines harboring homozygous deletions on the chromosome 10q23 region by two groups (Li et al., 1997; Steck et al., 1997) and by a group searching for the novel tyrosine phosphatases (Li and Sun, 1997). A series of PTEN/MMAC1 mutations were then identi®ed in sporadic tumors and cancer cell lines from various tissues including brain, endome- trium, prostate, breast, thyroid, and melanoma (Ri- singer et al., 1997; Cairns et al., 1997; Rhei et al., 1997; Dahia et al., 1997; Guldberg et al., 1997; Teng et al., 1997). Germline mutations of PTEN/MMAC1 were found to be associated with two inherited cancer predisposition disorders, Cowden's Syndrome (CS) (Liaw et al., 1997; Tsou et al., 1997, 1998; Nelen et al., 1997; Lynch et al., 1997) and Bannayan-Zonana Syndrome (BZS) (Marsh et al., 1997). Functionally, PTEN/MMAC1 protein is a dual-speci®city phospha- tase and has a high degree of substrate speci®city for the acidic peptide polyGlu 4 Tyr 1 (Li et al., 1997; Myers et al., 1997). The loss of phosphatase activity has been associated with a subgroup of missense mutations derived from both germline and somatic origin (Myers et al., 1997), and the catalytically inactive PTEN/ MMAC1 alleles (Furnari et al., 1997). Overexpression of the PTEN/MMAC1 gene in 3T3 cells showed an ability to inhibited cell migration by interacting with the focal adhesion kinase (FAK) (Tamura et al., 1998). In Taiwan, HCC is the leading cancer in males and third leading cancer in females. The level of AFB 1 urinary metabolites was correlated positively with the incidence of developing HCC in Taiwan (Chen et al., 1996). Most of the HCC cases were also chronic carriers *Correspondence: HC Tsou, Department of Dermatology, Columbia Presbyterian Hospital, 630 West 168th Street, VC1526, New York, New York 10032, USA The ®rst three authors contributed equally to this work Received 16 September 1998; revised 28 October 1998; accepted 5 January 1998 Oncogene (1999) 18, 3181 ± 3185 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc